Heterotrimeric G proteins couple cell surface receptors with intracellular effectors at the cytoplasmic face of membranes. The heterotrimer consists of a subunits which bind the guanine nucleotide and bg subunits which are tightly bound together. The membrane localization of G proteins is critical for their ability to relay signals between membrane-bound receptors and effectors. The a subunits undergo the post- translational modifications of myristoylation and palmitoylation which are important for their membrane attachment. Myristoylation, the addition of a 14 carbon fatty acid to the amino-terminal glycine, is critical for the membrane localization of a subset of a subunits primarily because it increases the affinity of the a subunit for the bg complex. Palmitoylation is the reversible addition of the 16 carbon palmitic acid to cysteine residues through a thioester bond. We found that mutation of the cysteine at residue 3 for as and ai prevents palmitoylation. Activation of the as subunit by isoproterenol or cholera toxin led to an increase in the level of palmitoylation. We found that palmitoylation functions to keep the as subunit at the membrane when it is free of the bg subunits. Palmitoylation requires that the subunit is membrane localized but does not require myristoylation. The bg complex can regulate the turnover of palmitate on the a subunits possibly by changing the availability of the cysteine for the modification. These studies on the membrane localization of G proteins enhance our understanding of the regulation and specificity involved in G protein- mediated signal transduction.